UC-NRLF 


3DS 


EXCHANGE 


. 


A  Study  of  the  Highly  Unsaturated  Fatty  Acids 
Occurring  in  Fish  Oils 


BY 


JOHN  BERNIS  BROWN 

B.S.,  University  of  Illinois,  1915. 
M.S.,  University  of  Illinois,  1917. 


THESIS 

hibmitted  in  Partial  Fulfillment  of  the  Requirements  for   the 

Degree  of 

DOCTOR  OF  PHILOSOPHY 
IN  CHEMISTRY 

IN 

THE  GRADUATE  SCHOOL 
OF  THE 

UNIVERSITY  OF  ILLINOIS 
1921 


EASTON,  PA.: 

ESCHBNBACH    PRINTING  COMPANY 
1920 


A  Study  of  the  Highly  Unsaturated  Fatty  Acids 
Occurring  in  Fish  Oils 


BY 


JOHN  BERNIS  BROWN 

B.S.,  University  of  Illinois,  1915. 
M.S.,  University  of  Illinois,  1917. 


THESIS 

Submitted  in  Partial  Fulfillment  of  the  Requirements  for   the 

Degree  of 
DOCTOR  OF  PHILOSOPHY 

IN  CHEMISTRY 

IN 

THE  GRADUATE  SCHOOL 
OF  THE 

UNIVERSITY  OF  ILLINOIS 
1921 


EASTON,  PA.: 

ESCHENBACH    PRINTING  COMPANY 
1920 


EXCHANGE 


ACKNOWLEDGMENT 

The  writer  wishes  to  express  his  great  indebtedness  to  Professor  G.  D. 
Beal  under  whose  able  direction  the  preparation  of  this  thesis  has  been 
carried  out,  and  who  has  made  many  valuable  suggestions  as  to  methods 
.of  attacking  the  problems  involved.  The  writer  desires  also  to  thank 
Professor  Roger  Adams  and  Doctor  C.  S.  Marvel  of  the  Division  of  Organic 
Chemistry  for  very  helpful  advice  concerning  a  number  of  the  organic 
chemical  problems  involved  in  the  thesis,  Mr.  Paul  Anders  for  construct- 
ing the  special  fractionating  apparatus  used,  and  Doctor  A.  M.  Patterson, 
former  Editor  of  Chemical  Abstracts,  for  offering  valuable  suggestions 
concerning  the  nomenclature  of  the  highly  unsaturated  acids. 


THE   HIGHLY   UNSATURATED   FATTY   ACIDS    OF   FISH    OILS 

Introduction 

While  engaged  in  the  preparation  of  pure  fatty  acids  for  use  in  medical 
research  during  the  late  war  under  the  auspices  of  the  National  Research 
Council,  the  writers  had  occasion  to  prepare  pure  clupanodonic  acid, 
CisHjsOj.  The  information  contained  in  the  literature  regarding  this 
acid  was  vague,  and  a  number  of  preliminary  studies  were  made  in  order 
to  divise  a  cheap  and  convenient  method  for  its  preparation.  The  method 
finally  adopted  was  a  modification  of  that  of  Riedel.1  The  methyl  esters, 
which  were  prepared  by  debromination  with  zinc  of  the  polybromides  of 
the  methyl  esters  of  menhaden  oil,  had  a  molecular  weight  too  high  for 
methyl  clupanodonate,  and  furthermore  distilled  over  too  wide  a  range 
to  be  a  pure  compound.  These  facts  indicated  that  we  were  dealing  with 
1  Riedel,  Ger.  pat.,  266,350. 


some  acids  other  than  clupanodonic.  Since  practically  none  of  the  prop- 
erties of  clupanodonic  acid  has  been  described,  the  investigation  was  con- 
tinued with  the  idea  of  determining  the  nature  of  the  pure  acid,  and  it  has 
finally  developed  into  an  attempt  at  a  characterization  of  the  highly 
unsaturated  acids  of  fish  oils. 

Hofstaedter  in  18542  was  the  first  to  discuss  in  detail  the  presence  of  an  unsaturated 
acid  in  a  marine  oil.  Investigations  by  Fahrion,3  Bull,4  Tolman,6  Twitchell6  and  others 
have  demonstrated  beyond  question  the  presence  of  highly  unsaturated  acids  in  both 
fish  and  marine  animal  oils.  By  brominating  the  acids  from  Japanese  sardine  oil  and 
reducing  the  resulting  bromides  with  zinc  and  alcoholic  hydrochloric  acid,  Tsujimoto7 
obtained  an  acid  with  an  iodine  number  of  344,  which  he  called  clupanodonic  acid. 
Majida  and  Okada8  reported  that  the  acids  obtained  in  this  fashion  had  a  molecular 
weight  too  high  for  clupanodonic  acid,  and  that  on  hydrogenation  they  obtained  an  ap- 
parent mixture  of  arachidic  and  behenic  acids. 

When  the  problem  had  been  under  way  for  18  months,  we  read  the  interesting  pre- 
liminary report  of  Tsujimoto  in  which  he  describes  the  separation  of  the  acids  of  fish 
oils  by  means  of  variations  in  the  solubility  of  their  lithium  salts  in  acetone.9  From  the 
analysis  of  4  fish  oils  he  concluded  that  the  highly  unsaturated  acids  were  chiefly  C2oH32O2 
and  C22H34O2,  the  latter  of  which  he  proposes  to  call  clupanodonic  acid  instead  of  that  to 
which  he  had  formerly  given  the  name.10  In  view  of  the  fact  that  this  name  has  been 
used  for  the  compound,  CisH28O2,  for  sixteen  years,  we  prefer  to  retain  it  and  are  using, 
therefore,  the  following  nomenclature11  for  the  unsaturated  acids  throughout  this  paper. 

Formula     Double  bonds      Acid  name  Formula     Double  bonds     Acid  name 

Ci6H26O2  3         Hexadecatrienoic  C20H3oO2  5         Bicosapentenoic 

CisH3oO2  3         Linolenic  C22H34O2  5         Docosapentenoic 

CisH28C2  4         Clupanodonic  C22H32O2  6         Docosahexenoic 

C20H32O2  4         Arachidonic 

Experimental  Part 

Attempted  Preparation  of  Clupanodonic  Acid  from  Menhaden  Oil 
Preparation  and  Bromination  of  the  Esters  of  Menhaden  Oil. — In 
order  to  compare  the  methyl,  ethyl  and  butyl  esters  of  menhaden  oil  and 
2  Hofstaedter,  Ann.,  91,  177  (1854). 
'  Fahrion,  Chem.-Ztg.,  17,  521  (1893). 
4  Bull,  ibid.,  23,  996  (1899);  Tidskrift  Kemi,  Farm.  Terapi,  14,  1  (1917). 

6  Tolman,  J.  Ind.  Eng.  Chem.,  1,  341  (1909). 

8  Twitchell,  ibid.,  6,  564  (1914);  9,  581  (1917). 

7  Tsujimoto,  J.  Coll.  Eng.  Imp.  Univ.  Tokyo,  4,  1  (1906). 

8  Majida  and  Okada,  Science  Repts.,  Tohoku  Imp.  Univ.,  3,  1  (1914). 

9  Tsujimoto,  /.  Chem.  Ind.  (Japan),  23,  1007  (1920). 

10  Since  the  above  was  written,  Professor  Tsujimoto,  writing  of  Japanese  sardine 
oil,    has  again  given  the  name  clupanodonic  acid  to  the  compound  C22H34O2  [  Chem. 
Umschau  Fette,  Oele,  Wachse  Harz,  33,  261  (1922)].     Since  the  acid  Ci8H28O2t  to  which 
the  name  was  originally  given,  undoubtedly  occurs  in  oils  from  the  herring  family,  we 
can  see  no  reason  for  the  change  in  the  use  of  the  name.     We  feel  that  more  confusion 
will  be  caused  by  a  change  now,  since  a  number  of  authors,  among  others  MacArthur 
and   Burton    [J.  Am.  Chem.  Soc.,  38,  1375   (1916)]  have  reported  clupanodonic  acid, 
Ci8H28O2,  as  a  constituent  of  animal  tissues. 

11  Dr.  A.  M.  Patterson  has  kindly  verified  the  nomenclature  of  these  acids. 


their  behavior  towards  bromination,  they  were  prepared  by  a  modified 
form  of  Haller's  method12  of  alcoholysis. 

Menhaden  oil  was  refluxed  with  1.5  times  its  weight  of  absolute  methyl  alcohol  or  an 
equivalent  amount  of  the  other  alcohols,  containing  about  2%  of  dry  hydrogen  chloride, 
for  24  hours.  The  mixture  was  cooled  and  washed  with  strong  brine  to  remove  the  ex- 
cess of  alcohol,  the  liberated  glycerol  and  hydrochloric  acid,  the  esters  then  being  distilled 
under  reduced  pressure.  The  esters  were  dissolved  in  9  times  their  weight  of  dry 
ether  and  bromine  slowly  added  in  10%  excess,  while  the  mixture  was  kept  at  a  temper- 
ature of  — 10°  to  — 5°  by  a  bath  of  ice  and  salt.  After  standing  for  6  hours  the  precipi- 
tated bromides  were  washed  with  ether  by  decantation  until  freed  from  bromine,  then 
dried  in  air.  These  methods  of  esterification  and  bromination  were  used  generally 
throughout  this  investigation.  The  percentage  yield  of  bromides  based  upon  the  original 
weight  of  ester,  or  polybromide  number,  and  the  percentage  of  bromine  in  the  bromides 
were  determined.  The  mixed  butyl  esters  of  the  acids  of  linseed  oil  were  carried  through 
a  like  process  to  determine  whether  evidences  of  substitution  would  appear. 

Determination  of  Bromine  in  the  Polybromides. — The  bromine  content 
of  the  ester  polybromides  was  determined  by  the  aid  of  the  Parr  peroxide 
bomb,  using  the  authors'  modification  of  the  procedure  outlined  by  Lemp 
arid  Broderson.13 

A  sample  of  about  0.3  g.  was  placed  in  the  fusion  cup  together  with  1  g.  of  accelerator, 
in  this  case  sodium  nitrate,  0.5  g.  of  granulated  sugar  and  a  measure  of  sodium  peroxide. 
After  ignition,  either  by  a  Bunsen  burner  or  the  improved  electrical  method,  the  fusion 
mixture  was  cooled  and  dissolved  in  150  cc.  of  water.  The~solution  was  acidified  with  22 
cc.  of  cone,  nitric  acid,  an  excess  of  0.1  N  silver  nitrate  solution  added  and  the  solution 
heated  to  boiling;  10  cc.  of  a  4%  solution  of  hydrazine  sulfate  was  added  to  reduce  any 
bromate  ions  present  and  boiling  continued  until  the  precipitate  had  coagulated  thor- 
oughly. After  the  mixture  had  boiled,  5  cc.  of  a  saturated  solution  of  ferric  alum  was 
added  and  the  excess  of  silver  nitrate  titrated  with  0.05  N  ammonium  thiocyanate  solu- 
tion. A  blank  determination  was  made  with  the  reagents  and  sugar  alone. 

TABLE  I 

A  COMPARISON  OF  THE  BEHAVIOR  OF  THE  METHYL,  ETHYL  AND  BUTYL  ESTERS  OF 
MENHADEN  OIL  TOWARDS  BROMINATION 

Boiling  Wt.          *  Wt.  Calc.  for 

point  ester      bromides    Polybro-      Bromine      clupano- 

Ester  at  15  mm.         G.  G.          mide  No.         %  donate 

Menhaden  Oil 

Methyl 195-240°       2100        805        38.3        68.31         68.79 

Ethyl* 195-240  600         195         32.5         67.43         67.84 

Ethyl 195-240         1270         430         33.8        67.48        67.84 

Ethyl 240-265          409         367         85.4        69.49         67.84 

w-Butyl 190-245          305          96        31 .5        67.64        65.82 

Linseed  Acids 

n-Butyl 190-240  100          51         51 .0        59.30        59.38* 

a  Two  lots  of  ethyl  esters  were  made  and  distilled  into  2  fractions  as  shown  above. 

The  higher-boiling  fractions  were  combined. 

6  59.38%  is  the  theoretical  bromine  content  of  butyl  hexabromo-stearate.     This 

compound  sintered  at  157°  and  melted  at  160°.     It  has  not  been  previously  described. 

12  Haller,  Compt.  rend.,  146,  250  (1908). 

13  Lemp  and  Broderson,  J.  Am.  Chem.  Soc.,  39,  2069  (1917). 


It  has  been  suggested  that  sodium  bisulfite  solution  be  used  as  a  reducing  agent  in- 
stead of  the  hydrazine  sulfate.  This  must  be  added  and  the  excess  oxidized  with  potas- 
sium permanganate  solution  before  the  addition  of  the  silver  nitrate  solution.  The 
results  with  this  procedure  were  not  always  of  the  best.  One  of  us  (J.  B.  B.)  has  had  an 
illuminating  experience  in  the  determination  of  iodine  by  this  method.  The  peroxide 
reaction  apparently  gave  only  iodate  since  silver  nitrate  in  the  acid  solution  formed  no 
precipitate.  Immediately  on  adding  the  hydrazine  sulfate  solution  silver  iodide  was 
precipitated  and  bubbles  of  nitrogen  gas  were  evolved.  There  was  no  appearance  of  free 
iodine.  When  sodium  bisulfite  was  used  as  the  reducing  agent  iodine  invariably  ap- 
peared. 

Was  Pure  Clupanodonic  Acid  Obtained? — The  percentage  of  bromine 
in  the  polybromides  obtained  from  the  higher-boiling  fraction  of  the  ethyl 
esters  and  from  the  butyl  esters  is  an  indication  of  the  presence  of  more 
highly  unsaturated  acids  than  clupanodonic.  These  acids  are  further- 
more of  higher  molecular  weight,  since  they  are  in  the  higher-boiling 
fraction.  Qualitative  tests  indicate  that  the  solubility  of  the  polybromides 
of  the  esters  increases  with  an  increase  in  the  molecular  weight  of  the 
alcohol  radical.  The  absence  of  more  than  traces  of  substitution  in  the 
method  of  bromination  employed  is  indicated  by  the  fact  that  butyl 
hexabromo-stearate  as  •  prepared  had  practically  the  calculated  bromine 
content,  and  no  evidence  of  formation  of  hydrobromic  acid  could  be  ob- 
tained. Denomination  ""of  the  bromides  of  the  fractions  of  the  lower- 
boiling  ethyl  ester  in  absolute  ethyl  alcohol  with  zinc  dust  gave  an  ester 
with  an  iodine  number  of  333.  The  acid  mixture  as  obtained  by  saponifi- 
cation  had  an  iodine  number  of  342  and  a  molecular  weight  by  titration 
of  305,  which  values  are  not  in  agreement  with  those  for  clupanodonic 
acid. 

The  Effect  of  Heat  on  the  Fatty  Acids  of  Menhaden  Oil.— The  free 
acids  of  fish  oils  could  be  distilled  only  with  difficulty,  since  a  thick  viscous 
liquid  remained  in  the  distilling  flasks.  To  test  the  effect  of  heat  a  quan- 
tity of  mixed  fatty  acids  from  menhaden  oil  was  placed  in  a  Claisen  flask 
and  heated  to  240°.  To  prevent  oxidation  a  gentle  stream  of  carbon 
dioxide  was  passed  over  the  acids.  Samples  were  withdrawn  from  time 
to  time  and  the  molecular  weight  was  determined  by  titration. 

TABUS  II 
THE  EFFECT  OF  HEAT  ON  THE  FATTY  ACIDS  OF  MENHADEN  OIL 

Time  of  heating,  min 00  60  105  165 

Mol.  wt 289.7  297.4  303.4  313.2 

The  maximum  temperature  to  which  the  fractions  are  subjected  under 
the  conditions  of  fractional  distillation  under  vacuum  is  240°.  A  similar 
experiment  conducted  with  the  esters  of  these  same  acids  showed  practically 
no  change  in  molecular  weight.  This  apparent  polymerization  may  ex- 
plain why  large  quantities  of  tarry  residue  are  found  in  the  distilling 
flask  on  fractionating  the  free  acids,  while  the  esters  may  be  distilled 


9 

almost  to  dryness.     All  subsequent  distillations,  when  possible,  were  of 
esters. 

Comparative  Analysis  of  Five  Commercial  Fish  Oils 
From  the  data  given  above  it  was  evident  that  in  the  sample  of  men- 
haden oil  used  there  were  present  highly  unsaturated  acids  other  than 
clupanodonic  acid.  Five  fish  oils  which  were  available  were  analyzed 
to  determine  the  probable  nature  of  the  highly  unsaturated  acids.  The 
constants  of  the  oils  are  given  in  Table  III. 

TABLE  III 
ANALYTICAL  CONSTANTS  OF  COMMERCIAL  FISH  OILS 

Oil  Sap.  No.  Iodine  No.  n«° 

Menhaden 191.2  151.7  1.4778 

Salmon 185.0  137.2  1.4768 

Cod 186.9  151.0  1.4770 

Herring 186.5  139.8  1.4765 

Sardine 187.3  158.1  1.4791 

One  kg.  of  each  oil  was  esterified  by  the  method  mentioned  previously 
and  the  methyl  esters  were  distilled  into  fractions  over  a  10°  range  under  15 
mm.  pressure.  In  the  case  of  menhaden  oil  3  kg.  was  used  and  the  dis- 
tillation repeated  thrice.  The  fractions  were  then  analyzed  for  free  acid, 
mean  molecular  weight  of  the  acids,  index  of  refraction,  iodine  number, 
polybromide  number  and  percentage  of  bromine  in  the  bromides,  while 
the  mean  molecular  weight  of  the  acids  was  calculated  from  the  molecular 
weight  of  the  esters  as  determined  by  saponification. 

The  fish  oils  examined  were  found  to  be  decidedly  similar  in  character. 
In  general  the  iodine  number,  polybromide  number  and  percentage  of 
bromine  in  the  bromides  increased  with  the  boiling  point,  the  main  di- 
vergence being  in  the  last  fraction,  which  might  easily  contain  decomposed 
material. 

The  oils  as  a  class  contain  acids  whose  esters  distil  at  much  too  high  a 
temperature  for  the  Ci8  series.  Among  these  higher  acids  are  those  which 
are  more  highly  unsaturated  than  clupanodonic  acid. 

Separation  of  the  Highly  Unsaturated  Acids 

From  the  facts  mentioned  above  it  is  quite  probable  that  the  clupano- 
donic acid  previously  described  in  the  literature  was  not  a  pure  compound, 
but  rather  a  mixture  of  a  number  of  acids.  Accordingly  a  number  of 
attempts  were  made  to  separate  the  fatty  acids  of  menhaden  oil,  the 
most  successful  procedure  being  applied  to  cod  and  herring  oils. 

Separation  by  Fractional  Distillation. — The  esters  from  3  kg.  of 
menhaden  oil  obtained  in  the  previous  experiment  were  fractionated  6  times. 
The  pressure  during  each  distillation  was  maintained  at  15  mm.  and  on 
the  last  distillation  the  fractions  were  collected  in  such  a  way  that  certain 


10 

ones  would  represent  as  far  as  possible  only  acids  of  the  same  carbon  con- 
tent. Although  the  intermediate  fractions  had  such  properties  as  to  indi- 
cate that  the  principal  fractions  were  not  chemical  individuals,  the  boiling 
point,  index  of  refraction,  molecular  weight  and  iodine  number  indicated 
the  predominance  in  each  of  these  fractions  of  acids  of  a  certain  group. 

TABLE  IV 

RESULTS  OF  ANALYSIS  OF  THE  FRACTIONS  FROM  THE  SIXTH  DISTILLATION  OF  THE  METHYL 
ESTERS  FROM  THREE  KILOGRAMS  OF  MENHADEN  OIL 

15  mm. 

range  Wt.  M.  m.  wt.  Iodine  Probable 

series 

Cl4 


C18 

C20 

C22 


From  the  series  of  analyses  just  tabulated  the  following  conclusions 
may  be  drawn:  (1)  It  is  possible  by  fractionation  of  the  methyl  esters  to 
make  a  rough  separation  of  acids  according  to  molecular  weight.  (2)  The 
important  acids  have  a  carbon  content  ranging  from  CH  to  C22.  Those  of 
the  lower  molecular  weights  are  largely  saturated.  (3)  The  iodine  numbers 
and  mean  molecular  weights  of  the  fractions  rise  rapidly  with  the  boiling 
points.  The  index  of  refraction  of  the  esters  was  also  found  to  increase 
regularly.  When  the  index  of  refraction  was  plotted  against  iodine  num- 
bers practically  a  straight  line  was  obtained,  the  main  divergence  being 
with  the  last  point.  The  same  general  relationship  is  seen  when  the 
index  of  refraction  is  plotted  against  molecular  weight,  the  breaks  at  the 
beginning  and  end  of  the  series  giving  the  graph  the  form  of  the  sign  of 
integration.  (4)  The  polybromide  number  of  the  esters  rises  with  the 
boiling  point,  while  the  bromine  content  of  the  derivatives  also  increases. 
Unsaturation  undoubtedly  increases  with  an  increase  in  molecular  weight. 

Separation  by  Metallic  Salts. — It  is  apparent  from  the  data  previously 
given  that  a  number  of  highly  unsaturated  acids  occur  in  fish  oils.  It 
is  furthermore  probably  true  that  no  one  has  succeeded  in  isolating  any 
of  them  in  the  free  state.  The  clupanodonic  acids  of  Tsujimoto  and  of 
Riedel,  previously  referred  to,  were  undoubtedly  complex  mixtures.  Var- 


Fraction 

°c. 

G. 

gii 

acids 

no. 

1 

156-166 

21.5 

1.4415 

234.1 

18.58 

2 

166-170 

26 

1.4408 

233.7 

14.73 

3 

170-180 

129 

1.4425 

239.5 

27.52 

4 

180-194 

257 

1.4470 

255.2 

53.2 

5 

194-198 

500 

1.4500 

260.5 

72.74 

6 

198-210 

208 

1.4546 

268.9 

101.8 

7 

210-216 

191 

1.4610 

276.6 

142.25 

8 

216-226 

237 

1.4670 

283.2 

185.2 

9 

226-230 

112 

1.4765 

293.7 

245.4 

10 

230-235 

66 

1.4810 

300.1 

277.0 

11 

235-240 

94 

1.4858 

305.1 

299.7 

12 

240-245 

107 

1.4895 

309.9 

306.5 

13 

245-250 

76 

1.4934 

317.2 

306.6 

14 

250-255 

31 

1.4960 

324.9 

304.1 

15 

255-260 

22 

1.4979 

326.4 

284.3 

11 

ious  methods  have  been  proposed  for  the  separation  of  the  saturated  and 
unsaturated  fatty  acids.  Most  of  the  methods  depend  upon  differences 
in  the  solubility  of  metallic  salts  of  the  unsaturated  acids  in  organic  sol- 
vents, those  of  the  unsaturated  acids  being  most  soluble.  Such  sepa- 
rations are  not  quantitative,  due  to  the  tendency  of  the  slightly  unsatur- 
ated acids  to  follow  the  saturated  acids  in  their  behavior,  and  also  to  an 
apparent  mutual  solubility  of  the  salts  of  the  two  types,  one  in  the  other. 
The  Lead  Soap-ether  Method. — Five  hundred  g.  of  refined  menhaden 
oil  (iodine  number  183.9;  saponification  number  191.8)  was  treated  ac- 
cording to  the  usual  procedure  for  the  lead  soap-ether  method  14>15  using 
only  such  modifications  as  were  necessitated  by  the  large  size  of  the  sample. 
The  lead  salts  so  separated  were  decomposed  as  usual  with-  hydrochloric 
acid  and  the  solid  and  liquid  acids  examined.  The  liquid  acids  weighed 
300  g.,  showed  an  average  molecular  weight  of  328.6  (the  abnormally  high 
value  is  probably  due  to  the  presence  of  a  small  amount  of  retained  ether) 
and  an  iodine  number  of  237.9;  whereas  the  solid  acids  weighed  150  g. 
and  showed  a  molecular  weight  of  264.6  and  an  iodine  number  of  55.3. 
The  liquid  acids  were  then  converted  into  then-  methyl  esters  and  dis- 
tilled under  15  mm.  pressure. 

TABLE  V 

THE  DISTILLATION  OF  THE  METHYL  ESTERS  OF  THE  UNSATURATED  ACIDS  OF  MENHADEN 
OIL  AS  OBTAINED  BY  THE  LEAD  SOAP-ETHER  METHOD 

Range  Wt. 

Fraction  °  C.  G.  Iodine  No.  « 

1  180-200  55  131.7  1.4560 

2  200-210  36  166.5  1.4622 

3  210-220  49  210.9  1.4683 

4  220-230  37  266.0  1.4771 

5  230-240  48  321.0  1.4860 

This  distillation  was  twice  repeated,  when  the  fraction  boiling  at  235- 
250°  had  an  iodine  number  of  334.1  and  a  molecular  weight  of  326.5. 
Since,  however,  the  lower  fractions  gave  evidence  of  the  presence  of  sat- 
urated acids  as  impurities  and  since  the  method  applied  to  large  quantities 
was  so  tedious,  further  investigation  by  this  procedure  was  discontinued. 

By  the  Barium  Soap-benzene  Method. — The  application  of  the  barium 
soap-benzene  method16  was  attempted.  This  is  based  on  the  solubility  of 
the  barium  soaps  of  fatty  acids  with  more  than  one  double  bond  in  cold 
benzene  containing  about  5%  of  95%  alcohol,  added  to  bring  a  trace  of 
moisture  into  the  benzene,  while  the  barium  salts  of  the  acids  of  the  oleic 
and  the  saturated  series  will  dissolve  in  the  hot  benzene-alcohol  but  crystal- 
lize on  cooling. 

14  Gusserow,  Ann.,  27,  153  (1828). 

16  Varrentrapp,  ibid.,  35,  197  (1840). 

16  Farnsteiner,  Z.  Nahr.  Genussm.,  [2]  1,  390  (1898). 


12 

The  sodium  soaps  of  menhaden  oil  were  neutralized  with  acetic  acid  and  precipi- 
tated in  the  cold  with  an  excess  of  barium  chloride  solution.  The  soaps  so  precipitated 
were  filtered  on  a  Biichner  funnel,  and  when  transferred  to  a  large  flask  and  heated 
on  a  steam-bath  for  a  few  minutes  so  agglutinated  and  shrunk  that  practically  all  of 
the  occluded  water  could  be  decanted.  The  soaps  were  then  treated  with  the  benzene- 
alcohol  mixture  and  refluxed  for  30  minutes  on  a  steam-bath.  The  solvent  was  poured  off 
and  the  residual  soaps  were  treated  with  a  fresh  portion  of  the  solvent.  This  treatment 
was  repeated  until  the  soaps  were  completely  disintegrated  and  practically  all  dissolved. 
The,  combined  solutions  were  then  allowed  to  cool  overnight,  and  filtered  from  the  crys- 
talline precipitate.  About  8  liters  of  solvent  was  required  for  500  g.  of  the  oil.  The 
acids  from  both  filtrate  and  precipitate  were  recovered  by  decomposing  the  soaps  with 
hydrochloric  acid  and  distilling  the  benzene.  The  last  traces  of  benzene  were  removed 
by  heating  in  a  vacuum.  The  results  obtained  in  the  first  trial  distillation  of  500  g.  were 
so  satisfactory  that  3  additional  distillations  were  made,  the  products  being  combined 
for  analysis. 

No.  of 

distillation  Character  G.  Mol.  wt.  no.  «20 

1  Liquid  260  306.0  267.0  1.4845 

2-3-4 

1 


Wt. 

Iodine 

Character 

G. 

Mol.  wt.                  no. 

Liquid 

260 

306.0            267.0 

1 

Liquid 

790 

305.9            261.4 

Solid 

144 

281.6              53.7 

Solid 

420 

Constants  not  determined 

One  kg.  of  these  liquid  acids  was  converted  into  their  methyl  esters  and  distilled 
thrice  under  15  mm.  pressure.  A  special  Claisen  flask  was  made  of  Pyrex  glass  for  this 
distillation  by  Mr.  Paul  Anders.  The  flask  had  an  especially  wide  neck  and  side  tube,  to 
prevent  the  liquid  from  bumping  over,  and  the  side  arm  was  inserted  a  short  distance  into 
the  upright  side  tube  in  order  to  prevent  esters  condensing  on  the  stopper  from  running 
into  the  receiver.  The  side  arm  was  also  especially  long  so  that  it  might  be  surrounded 
by  a  small  Pyrex  condenser  while  it  conducted  the  distillate  directly  into  a  Raikow  re- 
ceiver which  permitted  five  fractions  to  be  taken  without  breaking  the  vacuum.  The 
rubber  stoppers  were  protected  from  the  hot  ester  vapors  by  pinning  a  thin  sheet  of  cork 
over  their  lower  surfaces. 

TABLE  VI 

ANALYTICAL  CONSTANTS  OF  THE  METHYL  ESTERS  OF  THE  LIQUID  ACIDS  OF  MENHADEN 
OIL  OBTAINED  BY  THE  BARIUM  SOAP  SEPARATION,  AFTER  THREE  DISTILLATIONS 

Range 

(15  mm.)  Wt.  M.  m.  wt.  Iodine  no. 

Fraction  °  C.  G.  acids  of  ester  «18 

1  170-180  8  ...  ...  1.4627 

2  180-200  98  255.2  111.4  1.4536 

3  200-210  100  257.9  142.2  1.4580 

4  210-220  147  260.7  181.0  1.4640 

5  220-225  49  ...  231.2  1.4718 

6  225-230  86  ...  249.3  1.4740 

7  230-235  66  ...  280.1  1.4791 

8  235-240  95  304.0  316.5  1.4845 

9  240-245  65  308.5  334.6  1.4890 

10  245-250       43       312.6       347.7       1.4930 

11  250-255       58       321.6       348.4       1.4960 

12  255-260       34       336.7       330.9       1.4980 

Fractions  8-12,  boiling  over  5°  ranges  from  235—260°,  had  mean  molec- 
ular weights  ranging  from  304  to  336  and  iodine  numbers  for  the  esters 


13 

ranging  from  316  to  348.  Fraction  8  gave  values  corresponding  to  the 
theoretical  values  for  the  methyl  ester  of  arachidonic  acid.  Probably  the 
higher-boiling  fractions  contain  docosapentenoic  and  docosahexenoic  acids. 
The  polybromide  numbers  of  Fractions  8  and  11  were  determined  by 
the  following  procedure. 

Place  1-2  g.  of  the  ester  in  a  weighed  50cc.  centrifuge  tube  and  add  35  cc.  of  an- 
hydrous ether.  Place  the  tube  in  a  cooling  bath  kept  below  0°  and  add  bromine,  with 
vigorous  stirring  to  a  distinct  excess  or  until  the  solution  is  colored  red.  Allow  the  tube 
and  contents  to  stand  in  an  icebox  overnight  in  the  dark,  centrifuge,  decant  the  ether, 
and  wash  the  precipitate  4  times  by  centrifuging  with  40cc.  portions  of  ether.  Dry  the 
tube  and  contents  at  60°  for  2  hours  and  weigh.  The  weight  of  bromides,  divided  by  the 
weight  of  ester  and  multiplied  by  100,  gives  the  "polybromide  number."  In  order  to  deter- 
mine the  nature  of  the  residual  acids  in  the  ether  nitrates,  they  were  shaken  with  sodium 
thiosulfate  solution  to  remove  the  excess  of  bromine,  then  dried  with  calcium  chloride, 
the  ether  was  allowed  to  evaporate  and  the  residue  finally  dried  in  a  vacuum  oven  at  60°. 

Fraction  8  showed  a  polybromide  number  of  97.90,  giving  69.61%  of 
bromine  in  the  bromides,  and  56.56%  of  bromine  in  the  ether-soluble 
portions,  while  for  Fraction  11  the  polybromide  number  was  101.13  and 
the  bromine  percentages  70.07  and  56.05  respectively. 

Fraction  8,  above,  could  not  be  pure  methyl  arachidonate  since  this  would 
yield  a  bromine  derivative  with  66.78%  of  bromine,  whereas  the  derivative 
formed  actually  contained  69.61%.  If  this  fraction  contained  a  con- 
siderable quantity  of  methyl  docosapentenoate,  which  gives  a  polybromide 
with  71.66%  of  bromine,  such  results  might  be  obtained.  However, 
the  substitution  of  this  acid  in  part  or  entirely  would  give  iodine  numbers 
which  would  be  much  higher  than  those  obtained.  It  is  more  probable 
that  the  fraction  is  a  mixture  of  methyl  arachidonate  and  methyl  doco- 
sapentenoate with  small  quantities  of  the  esters  of  less  unsaturated  acids. 

An  examination  of  the  ether-soluble  bromides  leads  to  one  of  two  con- 
clusions: either  the  liquid  bromides  are  not  completely  saturated  with 
bromine,  because  of  some  sort  of  steric  hindrance,  or  they  are  isomeric 
liquid  bromides  mixed  with  esters  of  the  saturated  acids  or  with  bromides 
of  less  unsaturated  acids. 

Separation  by  Reduction  of  the  Polybromide s. — The  method  used 
in  the  preparation  of  these  polybromides  was  essentially  the  same  as  that 
which  had  previously  been  employed. 

A  sample  of  commercial  menhaden  oil,  dark  reddish-amber  in  color,  was  available. 
-Two  portions  of  3  kg.  each  were  converted  into  their  methyl  esters.  The  first  lot  was  re- 
fluxed  with  the  acid-alcohol  mixture  for  12  hours  and  the  second  for  24  hours.  The 
yields  of  esters  on  distillation,  between  the  usual  range  of  190-250°  under  15  mm.  pres- 
sure were  2000  g.  of  ester  for  the  first  trial  and  2700  g.  for  the  second.  The  methyl  esters 
were  brominated  in  kilogram  quantities  in  ether  solution  at  a  temperature  which  was 
kept  below  0°,  as  has  been  previously  described.  In  all,  1772  g.  of  polybromides  was  ob- 
tained. Three  portions  of  the  esters  were  brominated  in  ether  containing  10%  of  glacial 
acetic  acid,  but  no  appreciable  difference  could  be  observed  in  either  the  yield  or  the 
percentage  of  bromine  taken  up. 


14 

Previous  experiments  have  been  rewarded  with  low  yields  when  the  polybromides 
were  reduced.2  This  was  thought  to  be  due  possibly  to  the  insolubility  of  the  reacting  sub- 
stances. Accordingly  normal  butyl  alcohol  and  benzyl  alcohol  were  tried  as  solvents, 
since  the  polybromides  appeared  to  be  much  more  soluble  in  these  liquids.  Boiling 
benzyl  alcohol  dissolves  the  polybromides  to  the  extent  of  100  g.  per  liter,  giving  a 
dark  colored  solution  from  which  the  white  polybromides  deposit  on  cooling. 

Reduction  in  Benzyl  Alcohol. — One  hundred  g.  of  the  polybromides  and  an  excess  of 
zinc  dust  were  placed  in  a  Pyrex  flask  with  about  200  cc.  of  benzyl  alcohol.  The  mixture 
was  carefully  heated,  but  as  soon  as  the  reaction  had  begun  it  progressed  violently,  and 
the  heat  developed  was  sufficient  to  break  the  flask.  A  second  lOOg.  portion  was  mixed 
in  the  dry  condition  with  100  g.  of  zinc  dust  and  added  in  small  portions  to  250  cc.  of 
benzyl  alcohol  boiling  in  a  500  cc.  flask  under  an  air  condenser.  No  heat  was  applied 
after  the  addition  of  the  first  portions  of  the  mixture,  the  heat  of  the  reaction  being  suffi- 
cient to  keep  the  mixture  refluxing  vigorously.  After  cooling,  the  mixture  was  a  very 
thick  viscous  liquid  from  which  nothing  could  be  separated.  It  was  thought  that  the 
benzyl  alcohol  had  possibly  entered  into  the  reaction. 

Reduction  in  Butyl  Alcohol. — A  mixture  of  the  polybromides  with  zinc  dust  was  re- 
fluxed  for  10  hours  in  the  presence  of  normal  butyl  alcohol,  the  alcohol  filtered  off  and  the 
residue  washed  twice  with  fresh  portions  of  butyl  alcohol.  After  the  alcohol  was  removed 
an  attempt  was  made  to  distil  the  residue,  but  nothing  passed  over  even  under  15  mm. 
pressure.  After  the  distillation  flask  had  been  removed  from  the  heat  for  5  minutes  a 
strongly  exothermic  reaction  set  in  which  resulted  in  the  formation  of  a  mass  of  tar  in  the 
flask. 

Reduction  in  Methyl  Alcohol. — Equal  parts  of  the  polybromides  and  zinc  dust  were 
refluxed  in  methyl  alcohol  for  48  hours,  the  alcoholic  layer  was  filtered  off  and  the  solid 
residue  washed  thrice  with  alcohol,  the  alcoholic  filtrates  being  combined  and  the  alcohol 
distilled  under  atmospheric  pressure.  The  ester-alcohol  mixture  remaining  was  agitated 
with  warm  dil.  hydrochloric  acid  to  remove  the  remaining  alcohol  and  to  decompose 
any  zinc  soaps  present.  The  ester  layer  was  then  separated  and  refluxed  for  12  hours 
with  twice  its  weight  of  absolute  methyl  alcohol  and  hydrochloric  acid,  under  the  usual 
conditions  for  methanolysis,  this  operation  being  made  necessary  by  the  formation  of  some 
free  acid  during  the  debromination.  The  esters  which  were  finally  recovered  were  dis- 
tilled under  15  mm.  pressure.  The  first  two  runs  of  500  g.  each  gave  a  total  of  137  g.  of  re- 
duced ester;  yield,  45%.  A  third  run  of  1050  g.  of  bromides  gave  163  g.  of  esters;  yield, 
50.9% ;  iodine  number  of  esters,  368.5;  mean  molecular  weight  of  acids,  307.9;  w20,  1.4910. 

The  esters  distilled  between  215°  and  250°  under  15  mm.  pressure.  There  was 
practically  no  residue  in  the  distilling  flask  after  these  esters  were  distilled,  a  fact  ac- 
counted for  by  the  absence  of  free  acid  carried  into  the  alcoholic  solution  as  zinc  salt. 

Comparison  of  the  Acids  of  Cod  and  Herring  Oils 

The  highly  unsaturated  esters  from  cod  and  herring  oils  were  prepared  in  a  similar 
manner;  1620  g.  of  the  methyl  esters  from  2000  g.  of  cod  oil  gave  480  g.  of  bromides,  con- 
taining 70.75%  of  bromine.  These  when  debrominated  gave  93  g.  of  esters ;  yield,  67.65% . 
1875  g.  of  esters  from  2200  g.  of  herring  oil  gave  517  g.  of  bromides,  with  a  bromine  con- 
tent of  69.29% .  These  yielded  83  g.  of  esters,  or  52.3% . 

The  highly  unsaturated  esters  of  menhaden  oil  were  distilled  thrice  under  diminished 
pressure,  while  those  of  cod  and  herring  oils  were  distilled  only  once,  on  account  of  the 
small  quantity  available.  For  the  second  and  third  distillations  of  the  menhaden  oil 
esters,  the  fractions  were  introduced  in  order  into  the  distilling  flask  by  means  of  a  sep- 
aratory  funnel,  the  stem  of  which  had  been  drawn  out  to  a  fine  tip.  All  of  the  temper- 
atures were  corrected  to  15  mm.  pressure,  including  the  stem  correction  for  the  ther- 
mometer. The  last  4  fractions  of  the  esters  of  cod  oil  showed  abnormal  values  for  all 
but  molecular  weight,  due  to  accidental  overheating.  The  analyses  follow. 


15 

TABLE  VII 

THE  CONSTANTS  OF  THE  FRACTIONATED  METHYL  ESTERS  OF  THE  HIGHLY  UNSATURATED 
FATTY  ACIDS  OF  MENHADEN,  COD  AND  HERRING  OILS,  PREPARED  BY  REDUCTION  OF  THE 

POLYBROMIDES 
Menhaden  oil,  distilled  thrice 


Fraction 

Range 
(15  mm.) 

wt. 

G. 

M.  m.  wt.     Iodine 
acids              no.                  «*• 

Poly- 
bromide 
no. 

Br 

Mixed 

esters  (215-250°) 

307 

.9 

368.5 

1 

.4910 

105. 

33 

69. 

40 

1 

below  203° 

9.5 

322 

.0 

260.6 

1 

.4753 

. 

2 

203-213 

30.0 

274 

.1 

334.8 

1 

.4800 

. 

3 

213-218 

16.0 

280 

.8 

348.8 

1 

.4860 

94. 

67 

68. 

02 

4 

218-223 

22.0 

284 

.7 

359.4 

1 

.4888 

96. 

62 

69. 

71 

5 

223-228 

47.0 

292 

.2 

363.8 

1 

.4907 

101. 

9 

69. 

78 

6 

228-233 

35.5 

298 

.1 

372.8 

1 

.4919 

101. 

0 

69. 

61  » 

7 

233-238 

33.0 

307 

.5 

376.3 

1 

.4950 

101. 

21 

70. 

45 

8 

238-243 

31.0 

315 

.8 

379.2 

1 

.4970 

97. 

56 

70. 

40 

9 

243-248 

17.0 

317 

.4 

373.2 

1 

.4980 

76. 

02 

70. 

48 

10 

248-255 

11.0 

324 

.7 

357.9 

1 

.4987 

64. 

90 

70. 

99 

Cod 

oil, 

distilled  once 

Mixed 

esters  (210-247°) 

298 

.0 

383.5 

1 

.4912 

125. 

8 

70. 

4 

1 

below  213° 

11.0 

279 

.4 

362.2 

1 

.4865 

104. 

0 

68. 

70 

2 

213-218 

11.0 

286 

7 

369.8 

1 

.4881 

109. 

8 

69. 

21 

3 

218-223 

18.0 

293 

.0 

380.0 

1 

.4895 

113. 

0 

69. 

56 

4 

223-228 

11.5 

303 

.3 

305.9 

1 

.4940 

.  .  . 

5 

228-233 

4.0 

307 

.3 

277.9 

1 

.4985 

.  .  . 

, 

.  . 

~ 

6 

233-250 

5.5 

313 

.0 

257.5 

1 

.4992 

.  .  . 

m 

.  . 

, 

7 

250-270 

10.0 

323 

.5 

232.5 

1 

.4993 

.. 

. 

Herring  oil,  distilled 

once 

Mixed 

esters  (215-250°) 

299 

.2 

373.9 

1.4910 

113. 

25 

69. 

66 

1 

below  213° 

6.0 

277 

.8 

354.0 

1 

.4860 

97. 

7 

68. 

99 

2 

213-218 

7.0 

283 

.4 

361.8 

1 

.4873 

101. 

1 

68. 

81 

3 

218-223 

9.0 

286 

.0 

366.3 

1 

.4888 

103. 

0 

69. 

05 

4 

223-228 

11.5 

294 

.0 

369.5 

'l 

.4900 

111. 

0 

69. 

45 

5 

228-2-33 

16.5 

299 

.0 

376.0 

1 

.4928 

106. 

5 

69. 

69 

6 

233-238 

11.0 

309 

.3 

379.4 

1 

.4960 

102. 

0 

70. 

12 

7 

238-245 

4.5 

315 

.0 

372.0 

1 

.4992 

94. 

0 

70.61 

Menhaden 

oil, 

faction  2, 

redistilled 

2a 

180-195 

9.0 

266 

.8 

316.5 

1 

.4800 

72. 

78 

68. 

59 

2b 

195-205 

7.5 

274 

.5 

333.7 

1 

.4829 

.  . 

.  . 

2c 

205-212 

5.5 

282 

.7 

344.7 

1 

.4896 

. 

.  . 

.' 

The  Nature  of  the  Fatty  Acid 

The  following  conclusions  may  be  drawn  from  the  examination  of  the 
ester  fractions  of  the  various  oils  in  the  preceding  table. 

The  highly  unsaturated  acids  would  seem  to  contain  16,  18,  20  and  22 
carbon  atoms,  the  molecular  weights  of  the  tetra-unsaturated  acids  being 
248,  276,  304  and  332,  respectively.  The  lowest  mean  molecular  weight 
determined  was  266.8  (see  below),  which  is  9  points  lower  than  that  for 


16 

clupanodonic  acid.  The  highest  value  found  was  324.7,  which  is  only 
7  units  below  that  for  the  C22  acid. 

From  the  examination  of  the  iodine  numbers  it  will  be  seen  that  up  to 
the  last  two  fractions  the  unsaturation  increases  with  the  boiling  point  and 
molecular  weight.  The  calculated  iodine  numbers  of  the  methyl  esters 
of  the  acids  which  may  be  present  are:  hexadecatrienoate,  288.5;  clupano- 
donate,  350.1;  arachidonate,  319.3;  eicosapentenoate,  401.7;  docosatetren- 
oate,  293.5;  docosapentenoate,  369.0;  docosahexenoate,  445.2.  Fraction 
2  when  slowly  distilled  into  3  sub-fractions  gave  a  fraction  having  an  iodine 
number  of  316.5  and  a  molecular  weight  for  its  acids  of  266.8,  which  data 
indicate  a  mixture  composed  of  2/3  methyl  clupanodonate  and  l/8  methyl 
hexadecatrienoate.  The  data  for  Fraction  3  are  very  close  to  the  theoretical 
values  for  methyl  clupanodonate.  The  data  for  Fractions  6  and  7  indicate 
mixtures  of  methyl  arachidonate  and  eicosapentenoate,  while  those  for 
Fraction  9  indicate  methyl  docosatetrenoate,  docosapentenoate  and 
docosahexenoate . 

The  percentages  of  bromine  in  the  polybromides  of  the  various  fractions 
show  that  the  degree  of  unsaturation  increases  with  an  increase  in  the 
boiling  point  of  the  fraction  and,  therefore,  with  the  molecular  weight. 
One  of  the  sub-fractions  obtained  from  Fraction  2  and  also  Fraction  3 
yielded  bromides  with  68.59  and  68.6%  of  bromine  which  are  almost  in 
exact  agreement  with  the  calculated  value  for  methyl  octobromostearate. 
This  compound  has  not  been  previously  prepared.  It  is  a  white  amor- 
phous solid  which  melts  at  240°,  uncorr. 

The  following  percentage  values  for  bromine  content  have  been  calcu- 
lated: octobromostearate,  68.79;  octobromo-arachidate,  66.78;  decabromo- 
arachidate,  71.66;  octobromobehenate,  64.90;  decabromobehenate,  69.90; 
dodecabromobehenate,  73.72.  Fractions  6  and  7  would  seem  from  this 
also  to  consist  of  methyl  arachidonate  and  eicosapentenoate,  while  Frac- 
tions 9  and  10  are  probably  chiefly  methyl  docosapentenoate.  Since 
Fraction  10  yields  a  bromide  which  contains  more  bromine  than  Fraction 
9,  the  former  undoubtedly  contains  some  of  the  methyl  docosahexenoate. 
This  is  the  principal  evidence  which  we  have  of  the  presence  of  this  acid. 

The  polybromide  numbers  of  the  fractions  furnish  little  evidence  of 
the  nature  of  the  fatty  acids.  The  very  important  fact  which  should  be 
observed  here  is  that  they  are  only  about  1/8  of  the  theoretical  value,  which 
is  always  in  the  vicinity  of  300. 

On  the  basis  of  the  analyses  of  Fraction  3,  it  is  believed  that  this  fraction 
is  almost  pure  methyl  clupanodonate.  It  is  further  believed  that  this 
represents  the  first  time  that  a  derivative  of  clupanodonic  acid  has  been 
prepared  in  a  reasonably  pure  state.  The  constants  of  methyl  clupano- 
donate based  on  this  sample  are,  iodine  number  (Wijs)  348.8;  b.  p.  (15 
mm.)  215°;  nzo,  1.4860.  The  octobromide  is  described  above. 


r 


The  data  in  Table  VII  for  cod  and  herring  oils  offer  no  evidence  to  con- 
tradict our  conclusions  based  on  the  more  complete  distillation  of  the 
esters  of  menhaden  oil,  but  rather  confirm  most  of  these  data.  The  poly- 
bromide numbers  of  the  mixed  esters  of  the  3  oils  are  105.3,  125.8  and  113.3, 
values  which  are  about  1/3  of  those  calculated. 

The  Nature  of  the  Bromination  Reaction 

A  low  yield  of  polybromides  upon  bromination  of  the  pure,  highly  uii- 
saturated  esters  of  linolenic  and  linolic  acids  has  been  reported  by  Rollett17 
and  Erdmann.18  The  most  satisfactory  explanation  of  the  liquid  bromides 
is  that  perhaps  they  are  isomeric  soluble  bromides  with  correspondingly 
different  properties.  To  furnish  further  light  on  this  problem  the  follow- 
ing experiment  was  carried  out.  Starting  with  menhaden  oil,  the  methyl 
esters  were  prepared  and  these  reduced  to  the  esters  of  the  unsaturated 
acids.  Then,  under  quantitative  conditions,  the  supposedly  pure  un- 
saturated acids  were  prepared  by  saponification  and  brominated,  and  both 
the  liquid  and  solid  bromides  weighed,  reduced  and  again  brominated. 
Suitable  analyses  were  made  at  each  step.  The  diagram,  Table  VIII, 

TABUS  VIII 

QUANTITATIVE  BROMINATION  AND  REDUCTION  OF  MENHADEN  Oil,  ESTERS 
1900  g.  of  menhaden  oil-*-          1337  g.  of  methyl  esters-*-        500  g.  of  polybromides 
(68.62%  of  bromine)—*-        47  g.  of  methyl  esters  — *•         40  g.  of  fatty  acids.     Iodine 
number,  360.5.     Molecular  weight,  304.2 

33.19  g.  brominated 
,/  \ 


23.37  g.  insol.  bromides  (69.53%  of  Br) 
polybromide  no.,  70.44 

I 

5.5  g.  of  fatty  acid0 
iodine  no.,  380.6 
mol.  wt.,  317 
brominated  2.56  g.      — 


crystallized  2  g. 
66.36%     mol.  wt.,  667 


sol.  bromides 
6.11  g. 
65.93%  of  Br 


insol.  bromides,  2.65  g 


polybromide  no.,  103.7 
70.38%  of  Br 


sol.  bromides 


liquid,  70  g. 
62.53%  of  Br 

reduced 

iodine  no.,  278.5 
mol.  wt.,  301 
3.12  g.  brominated 

I 

sol.  bromides 
60.76%  Br 


insol.  bromides  0.66  g. 
polybromide  no.,  21.3 
67.45%  Br 

0  The  acid  obtained  here  represents  that  from  an  additional  7  g.  of  solid  bromides 
from  the  same  point  in  a  parallel  experiment. 

shows  the  result  of  this  experiment.     Although  no  final  conclusion  may  be 
drawn  from  these  data,  the  following  suggestions  may  be  made. 

The  liquid  bromides  seem  to  contain  in  general  considerably  less  bromine 
than  the  solid.     When  reduced,  the  former  give  a  product  with  an  iodine 
17  Rollett,  Z.  physiol.  Ghent.,  62,  410,  421  (1909). 
»  Erdmann  and  Bedford,  Ber.,  42,  1324  (1909). 


18 

number  considerably  less  than  the  original  acid,  while  the  molecular 
weight  is  only  slightly  less.  These  in  turn  yield  only  a  small  amount  of 
solid  bromides  on  bromination.  The  low  bromine  content  of  the  liquid 
bromides  and  the  decrease  in  iodine  number  suggest  a  change  in  the  degree 
of  unsaturation. 

There  seem  to  be  four  possibilities  of  change  here.  (1)  One  double  bond 
may  have  been  saturated  during  the  reduction.  (2)  There  may  have 
been  a  rearrangement  leaving  one  of  the  unsaturations  in  a  position  where 
it  cannot  be  brominated,  since  it  is  known  that  an  «-/?  unsaturation  causes 
a  reduction  in  the  iodine  number  of  an  acid.  (3)  Oxidation  may  have  oc- 
curred. (4)  A  double  bond  may  have  disappeared  by  ring  formation  as 
from  a  1-5  di-ene  grouping.  The  second  and  fourth  possibilities  are  the 
'  most  reasonable.  In  support  of  the  second  it  has  been  noticed  repeatedly 
in  this  Laboratory  that  at  0°  bromine  is  not  added  to  the  unsaturated 
acids  with  the  same  ease  as  iodine  monochloride  or  monobromide  in  at- 
tempts at  the  determination  of  the  bromine  absorption  values  of  drying 
vegetable  and  fish  oils.  In  support  of  the  possibility  of  ring  formation 
we  may  recall  the  fact  that  when  the  highly  unsaturated  esters  of  cod  oil 
were  accidentally  overheated,  there  was  a  rapid  lowering  of  the  iodine  and 
polybromide  numbers  with  no  change  in  molecular  weight.  Bromination 
might  catalyze  a  similar  change. 

Whatever  may  be  the  answer  to  the  question  of  the  liquid  bromides, 
apparently  1/8  or  less  of  the  ester  brominated  goes  to  form  the  normal 
ether-insoluble  bromides,  while  the  other  2/3  forms  a  product  with  4  to 
8%  less  bromine,  which  is  soluble  in  ether.  The  liquid  bromides  on 
debromination  yield  a  product  from  which  a  smaller  proportion  of  poly- 
bromides  may  be  obtained  on  rebromination  in  ether  solution. 

Presumably  the  material  soluble  in  ether  after  bromination  consists 
of  two  portions:  the  solid  bromides  which  are  dissolved  in  the  ether  solu- 
tion of  the  liquid  bromides,  and  the  true  liquid  bromides,  either  isomeric 
with  the  others  or  formed  from  altered  unsaturated  acids. 

Summary 

The  methyl,  ethyl  and  normal  butyl  esters  of  the  acids  of  menhaden 
oil  and  their  bromination  products  have  been  prepared  and  analyzed. 
The  presence  of  more  highly  unsaturated  acids  than  clupanodonic  is  indi- 
cated. 

The  analysis  of  the  methyl  ester  fractions  from  5  fish  oils  indicates  the 
presence  of  acids  more  highly  unsaturated  than  clupanodonic. 

By  esterifying  menhaden  oil  and  fractionating  the  esters  6  times  by 
distillation  under  diminished  pressure,  collecting  16  fractions,  the  presence 
of  acids  with  16,  18,  20  and  22  atoms  of  carbon  is  indicated. 

The  unsaturated  acids  obtained  by  the  lead  soap-ether  and  barium  soap- 


19 

benzene  separations  have  been  esterified  and  fractionated  and  their  bromine 
addition  products  analyzed. 

The  ether-insoluble  bromine  addition  products  of  the  unsaturated  acids 
have  been  debrominated  and  fractionated  and  their  bromine  addition 
products  analyzed. 

We  believe  that  we  have  demonstrated  the  presence  in  fish  oils  of  myris- 
tic,  palmitic,  palmitolic  (hexadecatrienoic),  clupanodonic  (octodecate- 
trenoic),  arachidonic,  eicosapentenoic,  docosapentenoic  and  docosa- 
hexenoic  acids. 


VITA 

The  writer  of  this  thesis  received  his  high  school  education  in  the  Sterling 
Township  High  School,  Sterling,  Illinois  during  the  years  1907  to  1911. 
He  entered  the  University  of  Illinois  in  the  fall  of  1911  and  pursued  the 
usual  four  year  course  in  chemistry.  He  received  the  B.S.  degree  in 
1915  and  entered  the  Graduate  School  of  the  University  of  Illinois  the 
same  year.  He  was  a  graduate  assistant  in  chemistry  from  1915-1917, 
when  he  received  the  M.S.  degree.  He  was  assistant  in  chemistry  for  a 
short  time  in  the  fall  of  1917,  when  he  resigned  to  enter  the  army.  He 
was  commissioned  and  detailed  to  work  under  Prof.  G.  D.  Beal  on  Chemical 
War  Problem  No.  112,  National  Research  Council,  which  work  was  carried 
out  at  the  University  of  Illinois  the  last  three  months  of  1918  for  which 
residence  credit  in  the  Graduate  School  was  allowed.  From  January, 
1919,  to  the  spring  of  1920  he  held  the  position  of  full  time  assistant  in 
chemistry.  He  held  the  Dupont  Fellowship  in  chemistry  during  1920- 
1921. 

The  writer  is  the  author  of  "The  Composition  of  the  Body  Fat  of  the 
Common  Woodchuck  (Marmota  Monax),"  master's  thesis,  unpublished, 
and  "The  Preparation  of  the  Pure  Fatty  Acids  and  their  Cholesteryl 
Esters,"  a  report  on  Chemical  War  Problem  No.  112,  unpublished. 


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